The invention relates generally to modulating cell adhesion and to inhibiting the binding of fibrinogen and other proteins to blood platelets, and inhibiting the aggregation of blood platelets specifically to the gp IIb/IIIa fibrinogen receptor site. Fibrinogen is a glycoprotein present in blood plasma that participates in platelet aggregation and in fibrin formation. Platelets are cell-like anucleated fragments, found in the blood of all mammals, that also participate in blood coagulation. Interaction of fibrinogen with the IIb/IIIa receptor site is known to be essential for normal platelet function.
When a blood vessel is damaged by an injury or other causative factor, platelets adhere to the disrupted subendothethial surface. The adherent platelets subsequently release biologically active constituents and aggregate. Aggregation is initiated by the binding of agonists, such as thrombin, epinephrine, or ADP to specific platelet membrane receptors. Stimulation by agonists results in exposure of latent fibrinogen receptors on the platelet surface, and binding of fibrinogen to the glycoprotein IIb/IIIa receptor complex.
Attempts have been made to use natural products and synthetic peptides to determine the mechanism of adhesion and platelet aggregation. For example, Rouslahti and Pierschbacher in Science, 238, 491-497 (1987), describe adhesive proteins such as fibronectin, vitronectin, osteopontin, collagens, thrombospondin, fibrinogen, and von Willebrand factor that are present in extracellular matrices and in blood. The proteins contain the tripeptide arginine-glycine-aspartic acid (RGD) as their glycoprotein IIb/IIIa recognition site. These arginine-glycine-aspartic acid containing tripeptides are recognized by at least one member of a family of structurally related receptors, integrins, which are heterodimeric proteins with two membrane-spanning subunits. The authors state that the conformation of the tripeptide sequence in the individual proteins may be critical to recognition specificity.
Cheresh in Proc. Nat'l Acad. Sci. U.S.A., 84, 6471-6475, (1987), describes an Arg-Gly-Asp directed adhesion receptor expressed by human endothethial cells that is structurally similar to the IIb/IIIa complex on platelets but is antigenically and functionally distinct. This receptor is directly involved in endothelial cell attachment to fibrinogen, von Willebrand factor, and vitronectin.
Pierschbacher and Rouslahti, in J. of Biol. Chem., 262, (36), 17294-17298 (1987) hypothesized that the Arg-Gly-Asp sequence alone would be a sufficient signal for receptor recognition and binding and that, therefore, the conformation of the tri-peptide sequence would be determinative. Various synthetic peptides were produced and the authors concluded that the sterochemical conformation of Arg-Gly-Asp as influenced by enantiomeric substitutions or additions to this sequence significantly influenced receptor-ligand interaction. The authors further showed that cyclization of a decapeptide by forming a disulfide bridge between non-terminal residues Pen and Cys, rendered the peptide much less effective at inhibiting attachment to fibronectin.
In Proc. Nat'l Acad. Sci. U.S.A., 81, 5985-5988 (1984), the same authors describe tetrapeptide variants of the cell recognition site of fibronectin that retain attachment-promoting activity. Peptides having a tetrapeptide recognition site are described in U.S. Pat. Nos. 4,589,881 and 4,614,517. A number of large polypeptide fragments in the cell-binding domain of fibronectin have cell-attachment activity. For example, see U.S. Pat. Nos. 4,517,686, 4,661,111 and U.S. Pat. No. 4,578,079.
Ruggeri et al., Proc. Nat'l Acad. Sci. U.S.A., 83, 5708-5712 (1986) explore a series of synthetic peptides designed in lengths to 16 residues, that contain RGD and a valine attached to the aspartic acid residue of RGD that inhibit fibrinogen binding to platelets. See also Koczewiak et al., biochem. 23, 1767-1774 (1984); Ginsberg et al., J. Biol. Chem. 260(7), 3931-3936 (1985); and Haverstick et al., Blood 66(4), 946-952 (1985). Other inhibitors are disclosed in Eur. Pat. App. Nos. 275,748 and 298,820.
A number of low molecular weight polypeptide factors have been isolated from snake venom. These factors apparently have high affinity for the gp IIb/IIIa complex. For example, Huang et al., J. Biol Chem., 262, 16157-16163 (1987); Huang et al., Biochemistry, 28, 661-666 (1989) describe the primary structure of the venom trigramin which is a 72 amino acid polypeptide that contains the RGD subunit. Echistatin is another compound which has high affinity for the gp IIb/IIIa complex. This polypeptide contains 49 amino acids and has the RGD subunit and various disulfide bridges. Gan et al., J. Biol. Chem., 263, 19827-19832 (1988). See also, Dennis et al., Proc. Nat'l Acad. Sci. USA, 87, 2471-2475 (1989). However, these snake venom factors also have high affinity for other members of the adhesive protein receptor family including the vitronectin and fibronectin receptors so are not selective for the gp IIb/IIIa complex.
While it is known that the tripeptide sequence Arg-Gly-Asp is present in certain polypeptides that can duplicate or inhibit the cell attachment-promoting effects of fibronectin and vitronectin, the tripeptide Arg-Gly-Asp has low activity. At present, there is little understanding of how other amino acids coupled to this sequence influence binding specificity. U.S. Pat. No. 5,023,233, assigned to Merck & Co., Inc., discloses small cyclic hexapeptides which contain the sequence Arg-Gly-Asp and are useful platelet aggregation inhibitors. U.S. Pat. No. 5,037,808 discloses the use of indolyl platelet-aggregation inhibitors which are believed to act by antagonizing interactions between fibrinogen and/or extracellular matrix proteins and the platelet gp IIb/IIIa receptor. U.S. Pat. No. 5,037,808 discloses guanidino peptide mimetic compounds that retain an Asp residue which inhibit platelet aggregation. WO 9014103 describes the use of antibody-poly-peptide conjugates wherein said polypeptides contain the Arg-Gly-Asp (RGD) sequence.
WO 9111458 discloses the use of large cyclic peptides containing RGD flanked by proline residues which are platelet aggregation inhibitors. WO 9101331 discloses small cyclic platelet aggregation inhibitors which are synthetic cyclic pentapeptides containing the tripeptide sequence Arg-Gly-Asp and a thioether linkage in the cycle. Hartman et al., WO 9408962 discloses compounds which are effective for inhibiting platelet aggregation by inhibiting fibrinogen binding to the gp IIb/IIIa receptor site, e.g., 5-2-(4-Piperidinyl)ethyl oxyl!-2-indolecarbonyl-2(S)-phenylsulfonylamino-.beta.-alanine (compound II, FIG. 1B), and related compounds. U.S. Pat. No. 5,051,405 discloses the use of peptides and pseudopeptides such as N-amidino piperidine-3-carboxylglycyl-L-aspartyl-L-valine that inhibit platelet aggregation and thrombus formation in mammalian blood. EP 445 796 discloses linear compounds which can include internal piperazinyl or piperidinyl derivatives. EP 437 367 discloses linear polypeptide fibrinogen receptor antagonists. U.S. Pat. No. 5,265,812 discloses compounds of the structure R.sup.1 -A-(W)a-X-(CH.sub.2)b-(Y).sub.c -B-Z-COOR wherein R.sup.1 is a guanidino or amidino moiety and A and B are chosen from specific monosubstituted aryl or heterocyclic moieties.
U.S. Pat. No. 5,281,585 describes a series of 1,3 disubstituted piperidones which are useful as oral fibrinogen receptor antagonists. One such compound, 3(R)-2-Piperidin-4-yl ethyl-2-piperidone-1!acetyl-3(R)-methyl-.beta.-alanine; is specifically described on column 15, line 8, columns 35-40, and columns 63-67. The compound is one of the most potent of these piperidones, having an IC.sub.50 for inhibiting aggregation of about 30 nm. Following administration of 0.3 mg/kg, p.o. to the compound inhibited platelet aggregation between 90-100% during the first three hours. Inhibition decreased over time, dropping below 20% after 8 hours.
The present invention provides a novel fibrinogen receptor antagonist that has significant, extended oral activity and is, therefore, useful for the reasons stated herein. The antagonist inhibits platelet aggregation at about 100% over an entire 8 hour period. A number of serious diseases and disorders involve hyperthrombotic complications which lead to intravascular thrombi and emboli. Myocardial infarction, stroke, phlebitis and a number of other serious conditions create the need for a fibrinogen receptor antagonist having potent and long-lasting efficacy.